The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412？bp. The open reading frame contains 603？bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction. 1. Introduction Ras family small GTPases play essential roles in a variety of cellular responses including cell proliferation, differentiation, survival, transformation, and tumor development [1–4]. The family has approximately 20 members in mammals [5, 6], and as many as 36 Ras family genes have been identified in humans [7] with evolutionarily conserved orthologs in Drosophila, C. elegans, S. cerevisiae, S. pombe, Dictyostelium, and plants [8]. This family includes the classical Ras proteins (H-Ras, N-Ras, K4A-Ras, and K4B-Ras), the R-Ras proteins (R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3), the Rap proteins (Rap1A, Rap1B, Rap2A, and Rap2B), and the Ral proteins (RalA and RalB) [9]. The first identified classical Ras (hereafter simply referred to as Ras) proteins have been studied most intensively. It was shown that Ras transduces signals from receptor-type tyrosine kinases to downstream effectors and thereby controls the proliferation and differentiation of various cell types [10]. Ras proteins function as molecular switches and are controlled by a regulated GDP/GTP cycle. Guanine nucleotide exchange factors (GEFs, e.g., SOS and mCDC25/GRF) promote the formation of active, GTP-bound Ras, whereas GTPase-activating proteins (GAPs, p120 and NF1 GAP) promote
References
[1]
P. Sun, H. Watanabe, K. Takano, T. Yokoyama, J. I. Fujisawa, and T. Endo, “Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells,” Genes to Cells, vol. 11, no. 9, pp. 1097–1113, 2006.
[2]
R. B. Blasco, S. Francoz, D. Santamaría et al., “c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma,” Cancer Cell, vol. 19, no. 5, pp. 652–663, 2011.
[3]
S. García-Silva, O. Martínez-Iglesias, L. Ruiz-Llorente, and A. Aranda, “Thyroid hormone receptor β1 domains responsible for the antagonism with the ras oncogene: role of corepressors,” Oncogene, vol. 30, no. 7, pp. 854–864, 2011.
[4]
A. L. Kennedy, J. P. Morton, I. Manoharan et al., “Activation of the PIK3CA/AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis,” Molecular Cell, vol. 42, no. 1, pp. 36–49, 2011.
[5]
G. W. Reuther and C. J. Der, “The Ras branch of small GTPases: Ras family members don't fall far from the tree,” Current Opinion in Cell Biology, vol. 12, no. 2, pp. 157–165, 2000.
[6]
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.
[7]
J. Colicelli, “Human RAS superfamily proteins and related GTPases,” Science's STKE, vol. 2004, no. 250, p. RE13, 2004.
[8]
K. Wennerberg, K. L. Rossman, and C. J. Der, “The Ras superfamily at a glance,” Journal of Cell Science, vol. 118, no. 5, pp. 843–846, 2005.
[9]
M. Rosário, H. F. Paterson, and C. J. Marshall, “Activation of the Ral and phosphatidylinositol 3′ kinase signaling pathways by the Ras-related protein TC21,” Molecular and Cellular Biology, vol. 21, no. 11, pp. 3750–3762, 2001.
[10]
T. Gotoh, Y. Niino, M. Tokuda et al., “Activation of R-Ras by Ras-guanine nucleotide-releasing factor,” Journal of Biological Chemistry, vol. 272, no. 30, pp. 18602–18607, 1997.
[11]
S. M. Graham, A. D. Cox, G. Drivas, M. G. Rush, P. D'Eustachio, and C. J. Der, “Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation,” Molecular and Cellular Biology, vol. 14, no. 6, pp. 4108–4115, 1994.
[12]
A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, “The ras protein family: evolutionary tree and role of conserved amino acids,” Biochemistry, vol. 30, no. 19, pp. 4637–4648, 1991.
[13]
G. M. Bokoch and C. J. Der, “Emerging concepts in the Ras superfamily of GTP-binding proteins,” The FASEB Journal, vol. 7, no. 9, pp. 750–759, 1993.
[14]
S. M. Graham, S. M. Oldham, C. B. Martin et al., “TC21 and Ras share indistinguishable transforming and differentiating activities,” Oncogene, vol. 18, no. 12, pp. 2107–2116, 1999.
[15]
K. T. Barker and M. R. Crompton, “Ras-related TC21 is activated by mutation in a breast cancer cell line, but infrequently in breast carcinomas in vivo,” British Journal of Cancer, vol. 78, no. 3, pp. 296–300, 1998.
[16]
Y. Zhang, J. Chen, Z. Lv, Z. Nie, X. Zhang, and X. Wu, “Can 29 kDa rhGM-CSF expressed by Silkworm pupae bioreactor bring into effect as active cytokine through orally administration?” European Journal of Pharmaceutical Sciences, vol. 28, no. 3, pp. 212–223, 2006.
[17]
Y. Z. Zhang, J. Chen, Z. M. Nie et al., “Expression of open reading frames in silkworm pupal cDNA library,” Applied Biochemistry and Biotechnology, vol. 136, no. 3, pp. 327–343, 2007.
[18]
T. Schwede, J. Kopp, N. Guex, and M. C. Peitsch, “SWISS-MODEL: an automated protein homology-modeling server,” Nucleic Acids Research, vol. 31, no. 13, pp. 3381–3385, 2003.
[19]
T. Maniatis, E. F. Fritsch, and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1982.
[20]
U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970.
[21]
J. A. Falkner, M. Kachman, D. M. Veine, A. Walker, J. R. Strahler, and P. C. Andrews, “Validated MALDI-TOF/TOF mass spectra for protein standards,” Journal of the American Society for Mass Spectrometry, vol. 18, no. 5, pp. 850–855, 2007.
[22]
Q. Sheng, J. Xia, Z. Nie, and Y. Zhang, “Cloning, expression, and cell localization of a novel small heat shock protein gene: BmHSP25.4,” Applied Biochemistry and Biotechnology, vol. 162, no. 5, pp. 1297–1305, 2010.
[23]
L. Kong, Z. Lv, J. Chen et al., “Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori),” Biochimica et Biophysica Acta, vol. 1770, no. 12, pp. 1598–1604, 2007.
[24]
I. R. Vetter and A. Wittinghofer, “The guanine nucleotide-binding switch in three dimensions,” Science, vol. 294, no. 5545, pp. 1299–1304, 2001.
[25]
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.
