The Ras subfamily is the member of small G proteins superfamily involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation, and survival. Bombyx mori Ras-like protein (BmRas1) may belong to the Ras subfamily. It contained an H-N-K-Ras-like domain. The BmRas1 mRNA consisted of 1459？bp. The open reading frame contained 579？bp, encoding 192 amino acids. The protein had such secondary structures as α-helices, extended strand, and random coil. BmRas1 was expressed successfully in E. coli BL21. The recombinant protein was purified with metal-chelating affinity chromatography. The GTPase activity of purified protein was determined by FeSO4-(NH4)2MoO4 assay. The results showed that purified recombinant protein had intrinsic activity of GTPase. High titer polyclonal antibodies were generated by New Zealand rabbit immunized with purified protein. The gene expression features of BmRas1 at different stages and in different organs of the fifth instar larvae were analyzed by Western blot. The results showed that BmRas1 was expressed highly in three development stages including egg, pupae, and adult, but low expression in larva. BmRas1 was expressed in these tissues including head, malpighian tubule, genital gland, and silk gland. The purified recombinant protein would be utilized to further function studies of BmRas1. 1. Introduction Ras genes were first identified as homologues of rodent sarcoma virus genes. In 1982, human DNA sequences homologous to the transforming oncogenes of the v-Harvey (H-Ras) and Kirsten (K-Ras) rat sarcoma virus were identified in DNA sequences derived from a human bladder and a human lung cancer cell line, respectively. There are three mammalian Ras proteins: H-Ras, N-Ras, and K-Ras, which consisted of 188-189 amino acid (p21 proteins), encoded by three ras genes [1]. The Ras isoforms are highly homologous [2]. Ras proteins are positioned at the inner surface of the plasma membrane where they serve as binary molecular switches to transduce extracellular ligand-mediated stimuli into the cytoplasm to control signal transduction pathways that influence cell growth, differentiation, and apoptosis [3, 4]. The Ras protein is the prototype of the Ras superfamily of small GTPases, which share a high degree of sequence similarity and a common three-dimensional structure, called the GTP-binding domain. This domain enables them to act as molecular switches cycling between two defined conformational states: an inactive guanosine-diphosphate (GDP-) bound and an active guanosine-triphosphate-(GTP-) bound
References
[1]
C. J. Der, T. G. Krontiris, and G. M. Cooper, “Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kristen sarcoma viruses,” Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 11 I, pp. 3637–3640, 1982.
[2]
S. L. Campbell, R. Khosravi-Far, K. L. Rossman, G. J. Clark, and C. J. Der, “Increasing complexity of Ras signaling,” Oncogene, vol. 17, no. 11, pp. 1395–1413, 1998.
[3]
R. Khosravi-Far and C. J. Der, “The Ras signal transduction pathway,” Cancer and Metastasis Reviews, vol. 13, no. 1, pp. 67–89, 1994.
[4]
T. Satoh and Y. Kaziro, “Ras in signal transduction,” Seminars in Cancer Biology, vol. 3, no. 4, pp. 169–177, 1992.
[5]
M. S. Boguski and F. McCormick, “Proteins regulating Ras and its relatives,” Nature, vol. 366, no. 6456, pp. 643–654, 1993.
[6]
I. R. Vetter and A. Wittinghofer, “The guanine nucleotide-binding switch in three dimensions,” Science, vol. 294, no. 5545, pp. 1299–1304, 2001.
[7]
E. A. Elion, “Routing MAP kinase cascades,” Science, vol. 281, no. 5383, pp. 1625–1626, 1998.
[8]
H. R. Bourne, D. A. Sanders, and F. McCormick, “The GTPase superfamily: conserved structure and molecular mechanism,” Nature, vol. 349, no. 6305, pp. 117–127, 1991.
[9]
A. Ehrhardt, G. R. A. Ehrhardt, X. Guo, and J. W. Schrader, “Ras and relatives—job sharing and networking keep an old family together,” Experimental Hematology, vol. 30, no. 10, pp. 1089–1106, 2002.
[10]
A. D. Cox and C. J. Der, “Ras family signaling: therapeutic targeting,” Cancer Biology & Therapy, vol. 1, no. 6, pp. 599–606, 2002.
[11]
M. Wang and P. W. Sternberg, “Pattern formation during C. elegans vulval induction,” Current Topics in Developmental Biology, vol. 51, pp. 189–220, 2001.
[12]
J. T. Groves and J. Kuriyan, “Molecular mechanisms in signal transduction at the membrane,” Nature Structural and Molecular Biology, vol. 17, no. 6, pp. 659–665, 2010.
[13]
A. Rebollo and A. C. Martínez, “Ras proteins: recent advances and new functions,” Blood, vol. 94, no. 9, pp. 2971–2980, 1999.
[14]
A. Wittinghofer, “Signal transduction via Ras,” Biological Chemistry, vol. 379, no. 8-9, pp. 933–937, 1998.
[15]
M. E. Katz and F. McCormick, “Signal transduction from multiple Ras effectors,” Current Opinion in Genetics and Development, vol. 7, no. 1, pp. 75–79, 1997.
[16]
R. Treisman, “Regulation of transcription by MAP kinase cascades,” Current Opinion in Cell Biology, vol. 8, no. 2, pp. 205–215, 1996.
[17]
M. J. Robinson and M. H. Cobb, “Mitogen-activated protein kinase pathways,” Current Opinion in Cell Biology, vol. 9, no. 2, pp. 180–186, 1997.
[18]
H. J. Schaeffer and M. J. Weber, “Mitogen-activated protein kinases: specific messages from ubiquitous messengers,” Molecular and Cellular Biology, vol. 19, no. 4, pp. 2435–2444, 1999.
[19]
P. J. Casey and M. C. Seabra, “Protein prenyltransferases,” Journal of Biological Chemistry, vol. 271, no. 10, pp. 5289–5292, 1996.
[20]
J. A. Glomset and C. C. Farnsworth, “Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes,” Annual Review of Cell Biology, vol. 10, pp. 181–205, 1994.
[21]
J. H. Jackson, C. G. Cochrane, J. R. Bourne, P. A. Solski, J. E. Buss, and C. J. Der, “Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 8, pp. 3042–3046, 1990.
[22]
J. F. Hancock, A. I. Magee, J. E. Childs, and C. J. Marshall, “All ras proteins are polyisoprenylated but only some are palmitoylated,” Cell, vol. 57, no. 7, pp. 1167–1177, 1989.
[23]
P. J. Casey, P. A. Solski, C. J. Der, and J. E. Buss, “p21ras is modified by a farnesyl isoprenoid,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, no. 21, pp. 8323–8327, 1989.
[24]
Q. Xia, Z. Zhou, C. Lu et al., “A draft sequence for the genome of the domesticated silkworm (Bombyx mori),” Science, vol. 306, no. 5703, pp. 1937–1940, 2004.
[25]
L. Ma, Z. Hong, and Z. Zhang, “Perinuclear and nuclear envelope localizations of Arabidopsis Ran proteins,” Plant Cell Reports, vol. 26, no. 8, pp. 1373–1382, 2007.
[26]
C. Der and J. Gunzburg, “Ras family proteins,” in RAS Family GTPases, A. Ridley and J. Frampton, Eds., pp. 295–339, Springer, Amsterdam, The Netherlands, 2006.
[27]
H. Luo, P. E. Rose, T. M Roberts, and C. R Dearolf, “The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila,” Molecular Genetics and Genomics, vol. 267, no. 1, pp. 57–63, 2002.
[28]
R. M. Riley, W. Jin, and G. Gibson, “Contrasting selection pressures on components of the Ras-mediated signal transduction pathway in Drosophila,” Molecular Ecology, vol. 12, no. 5, pp. 1315–1323, 2003.
[29]
D. A. Baker, B. Mille-Baker, S. M. Wainwright, D. Ish-Horowicz, and N. J. Dibb, “Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila,” Nature, vol. 411, no. 6835, pp. 330–334, 2001.
[30]
R. Gasperini and G. Gibson, “Absence of protein polymorphism in the Ras genes of Drosophila melanogaster,” Journal of Molecular Evolution, vol. 49, no. 5, pp. 583–590, 1999.
[31]
E. Y. Mantrova and T. Hsu, “Down-regulation of transcription factor CF2 by Drosophila Ras/MAP kinase signaling in oogenesis: cytoplasmic retention and degradation,” Genes and Development, vol. 12, no. 8, pp. 1166–1175, 1998.
[32]
T. Ogura, A. Tan, T. Tsubota, T. Nakakura, and T. Shiotsuki, “Identification and expression analysis of ras gene in silkworm, Bombyx mori,” PLoS One, vol. 4, no. 11, article e8030, 2009.
[33]
K. Moriya, T. Tsubota, N. Ishibashi et al., “Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated,” Insect Molecular Biology, vol. 19, no. 3, pp. 291–301, 2010.
[34]
S. Eisenberg and Y. I. Henis, “Interactions of Ras proteins with the plasma membrane and their roles in signaling,” Cellular Signalling, vol. 20, no. 1, pp. 31–39, 2008.
