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. 2000 Feb 1;82(5):1035–1040. doi: 10.1054/bjoc.1999.1039

Decreased GTPase activity of K- ras mutants deriving from human functional adrenocortical tumours

S-R Lin 2, C-H Hsu 2, J-H Tsai 1, J-Y Wang 2, T-J Hsieh 2, C-H Wu 2
PMCID: PMC2374427  PMID: 10737386

Abstract

Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K- ras gene mutation. In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61). To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K- Ras oncogene and the alterations of its biocharacteristics, mutant K- Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV. Mutant K- Ras genes were expressed at high levels in Escherichia coli and the resultant K- Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. The purified K- Ras protein from E. coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase-activating protein for Ras. The results showed that the wild-type cellular K- Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K- Ras gene, which mutated at codon 60. With regards to the codon 15, 16, 18 and 31 mutant K- Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K- Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K- Ras protein. These results indicated that mutations at these hot spots of K- Ras gene were indeed activated K- Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove. © 2000 Cancer ResearchCampaign

Keywords: GTPase, K-ras mutants, functional adrenocortical tumours

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Selected References

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  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Ahmadian M. R., Wiesmüller L., Lautwein A., Bischoff F. R., Wittinghofer A. Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin. J Biol Chem. 1996 Jul 5;271(27):16409–16415. doi: 10.1074/jbc.271.27.16409. [DOI] [PubMed] [Google Scholar]
  3. Anker P., Lefort F., Vasioukhin V., Lyautey J., Lederrey C., Chen X. Q., Stroun M., Mulcahy H. E., Farthing M. J. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology. 1997 Apr;112(4):1114–1120. doi: 10.1016/s0016-5085(97)70121-5. [DOI] [PubMed] [Google Scholar]
  4. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  5. Boriack-Sjodin P. A., Margarit S. M., Bar-Sagi D., Kuriyan J. The structural basis of the activation of Ras by Sos. Nature. 1998 Jul 23;394(6691):337–343. doi: 10.1038/28548. [DOI] [PubMed] [Google Scholar]
  6. Caduff R. F., Svoboda-Newman S. M., Ferguson A. W., Johnston C. M., Frank T. S. Comparison of mutations of Ki-RAS and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors. Am J Surg Pathol. 1999 Mar;23(3):323–328. doi: 10.1097/00000478-199903000-00012. [DOI] [PubMed] [Google Scholar]
  7. Capon D. J., Seeburg P. H., McGrath J. P., Hayflick J. S., Edman U., Levinson A. D., Goeddel D. V. Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations. Nature. 1983 Aug 11;304(5926):507–513. doi: 10.1038/304507a0. [DOI] [PubMed] [Google Scholar]
  8. Chen Q., Lin T. H., Der C. J., Juliano R. L. Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras [corrected]. J Biol Chem. 1996 Jul 26;271(30):18122–18127. doi: 10.1074/jbc.271.30.18122. [DOI] [PubMed] [Google Scholar]
  9. Downward J. Control of ras activation. Cancer Surv. 1996;27:87–100. [PubMed] [Google Scholar]
  10. Feliciello A., Giuliano P., Porcellini A., Garbi C., Obici S., Mele E., Angotti E., Grieco D., Amabile G., Cassano S. The v-Ki-Ras oncogene alters cAMP nuclear signaling by regulating the location and the expression of cAMP-dependent protein kinase IIbeta. J Biol Chem. 1996 Oct 11;271(41):25350–25359. doi: 10.1074/jbc.271.41.25350. [DOI] [PubMed] [Google Scholar]
  11. Gao H. G., Chen J. K., Stewart J., Song B., Rayappa C., Whong W. Z., Ong T. Distribution of p53 and K-ras mutations in human lung cancer tissues. Carcinogenesis. 1997 Mar;18(3):473–478. doi: 10.1093/carcin/18.3.473. [DOI] [PubMed] [Google Scholar]
  12. Grendys E. C., Jr, Barnes W. A., Weitzel J., Sparkowski J., Schlegel R. Identification of H, K, and N-ras point mutations in stage IB cervical carcinoma. Gynecol Oncol. 1997 May;65(2):343–347. doi: 10.1006/gyno.1997.4649. [DOI] [PubMed] [Google Scholar]
  13. Gross M., Sweet R. W., Sathe G., Yokoyama S., Fasano O., Goldfarb M., Wigler M., Rosenberg M. Purification and characterization of human H-ras proteins expressed in Escherichia coli. Mol Cell Biol. 1985 May;5(5):1015–1024. doi: 10.1128/mcb.5.5.1015-1024.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  15. Hattori S., Maekawa M., Nakamura S. Identification of neurofibromatosis type I gene product as an insoluble GTPase-activating protein toward ras p21. Oncogene. 1992 Mar;7(3):481–485. [PubMed] [Google Scholar]
  16. Hughes P. E., Renshaw M. W., Pfaff M., Forsyth J., Keivens V. M., Schwartz M. A., Ginsberg M. H. Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway. Cell. 1997 Feb 21;88(4):521–530. doi: 10.1016/s0092-8674(00)81892-9. [DOI] [PubMed] [Google Scholar]
  17. Lautenberger J. A., Ulsh L., Shih T. Y., Papas T. S. High-level expression in Escherichia coli of enzymatically active Harvey murine sarcoma virus p21ras protein. Science. 1983 Aug 26;221(4613):858–860. doi: 10.1126/science.6308763. [DOI] [PubMed] [Google Scholar]
  18. Leitner J. W., Kline T., Carel K., Goalstone M., Draznin B. Hyperinsulinemia potentiates activation of p21Ras by growth factors. Endocrinology. 1997 May;138(5):2211–2214. doi: 10.1210/endo.138.5.5240. [DOI] [PubMed] [Google Scholar]
  19. Leon J., Guerrero I., Pellicer A. Differential expression of the ras gene family in mice. Mol Cell Biol. 1987 Apr;7(4):1535–1540. doi: 10.1128/mcb.7.4.1535-1540.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lim H. H., Michael G. J., Smith P., Lim L., Hall C. Developmental regulation and neuronal expression of the mRNA of rat n-chimaerin, a p21rac GAP:cDNA sequence. Biochem J. 1992 Oct 15;287(Pt 2):415–422. doi: 10.1042/bj2870415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lim L., Manser E., Leung T., Hall C. Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur J Biochem. 1996 Dec 1;242(2):171–185. doi: 10.1111/j.1432-1033.1996.0171r.x. [DOI] [PubMed] [Google Scholar]
  22. Lin S. R., Tsai J. H., Yang Y. C., Lee S. C. Mutations of K-ras oncogene in human adrenal tumours in Taiwan. Br J Cancer. 1998 Apr;77(7):1060–1065. doi: 10.1038/bjc.1998.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  24. Miao W., Eichelberger L., Baker L., Marshall M. S. p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues. J Biol Chem. 1996 Jun 28;271(26):15322–15329. doi: 10.1074/jbc.271.26.15322. [DOI] [PubMed] [Google Scholar]
  25. Molloy D. P., Owen D., Grand R. J. Ras binding to a C-terminal region of GAP. FEBS Lett. 1995 Jul 17;368(2):297–303. doi: 10.1016/0014-5793(95)00657-u. [DOI] [PubMed] [Google Scholar]
  26. Mulcahy H. E., Lyautey J., Lederrey C., qi Chen X., Anker P., Alstead E. M., Ballinger A., Farthing M. J., Stroun M. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res. 1998 Feb;4(2):271–275. [PubMed] [Google Scholar]
  27. Nur-E-Kamal M. S., Maruta H. The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP. Mol Biol Cell. 1992 Dec;3(12):1437–1442. doi: 10.1091/mbc.3.12.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Overbeck A. F., Brtva T. R., Cox A. D., Graham S. M., Huff S. Y., Khosravi-Far R., Quilliam L. A., Solski P. A., Der C. J. Guanine nucleotide exchange factors: activators of Ras superfamily proteins. Mol Reprod Dev. 1995 Dec;42(4):468–476. doi: 10.1002/mrd.1080420415. [DOI] [PubMed] [Google Scholar]
  29. Pells S., Divjak M., Romanowski P., Impey H., Hawkins N. J., Clarke A. R., Hooper M. L., Williamson D. J. Developmentally-regulated expression of murine K-ras isoforms. Oncogene. 1997 Oct 9;15(15):1781–1786. doi: 10.1038/sj.onc.1201354. [DOI] [PubMed] [Google Scholar]
  30. Porras A., Santos E. The insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells: dissociation between Raf-1 kinase and the MAPK/RSK cascade. Int J Obes Relat Metab Disord. 1996 Mar;20 (Suppl 3):S43–S51. [PubMed] [Google Scholar]
  31. Powers S., O'Neill K., Wigler M. Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):390–395. doi: 10.1128/mcb.9.2.390-395.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Santos E., Nebreda A. R. Structural and functional properties of ras proteins. FASEB J. 1989 Aug;3(10):2151–2163. doi: 10.1096/fasebj.3.10.2666231. [DOI] [PubMed] [Google Scholar]
  33. Segal M., Willumsen B. M., Levitzki A. Residues crucial for Ras interaction with GDP-GTP exchangers. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5564–5568. doi: 10.1073/pnas.90.12.5564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shirouzu M., Fujita-Yoshigaki J., Ito Y., Koide H., Nishimura S., Yokoyama S. A glutamic acid residue at position 31 of Ras protein is essential to the signal transduction for neurite outgrowth of PC12 cells and the stimulation of GTPase activity by GAPRas. Oncogene. 1992 Mar;7(3):475–480. [PubMed] [Google Scholar]
  35. Slamon D. J., Cline M. J. Expression of cellular oncogenes during embryonic and fetal development of the mouse. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7141–7145. doi: 10.1073/pnas.81.22.7141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sung Y. J., Carter M., Zhong J. M., Hwang Y. W. Mutagenesis of the H-ras p21 at glycine-60 residue disrupts GTP-induced conformational change. Biochemistry. 1995 Mar 14;34(10):3470–3477. doi: 10.1021/bi00010a040. [DOI] [PubMed] [Google Scholar]
  37. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  38. Winston L. A., Hunter T. JAK2, Ras, and Raf are required for activation of extracellular signal-regulated kinase/mitogen-activated protein kinase by growth hormone. J Biol Chem. 1995 Dec 29;270(52):30837–30840. doi: 10.1074/jbc.270.52.30837. [DOI] [PubMed] [Google Scholar]
  39. Wittinghofer A., Scheffzek K., Ahmadian M. R. The interaction of Ras with GTPase-activating proteins. FEBS Lett. 1997 Jun 23;410(1):63–67. doi: 10.1016/s0014-5793(97)00321-9. [DOI] [PubMed] [Google Scholar]
  40. Xu G. F., Lin B., Tanaka K., Dunn D., Wood D., Gesteland R., White R., Weiss R., Tamanoi F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov 16;63(4):835–841. doi: 10.1016/0092-8674(90)90149-9. [DOI] [PubMed] [Google Scholar]
  41. Yamashita N., Minamoto T., Ochiai A., Onda M., Esumi H. Frequent and characteristic K-ras activation in aberrant crypt foci of colon. Is there preference among K-ras mutants for malignant progression? Cancer. 1995 Mar 15;75(6 Suppl):1527–1533. doi: 10.1002/1097-0142(19950315)75:6+<1527::aid-cncr2820751524>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]

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