Cross talk between oncogene and ant-oncogene via the Crk oncogene product

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 10470064
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Experimental pathology
• Research Institution: Osaka University (2001); Research Institute, International Medical Center of Japan (1998-2000)
• Principal Investigator: MATSUDA Michiyuki Institute of Microbial Diseases, Tumor Viology, Professor, 微生物病研究所, 教授 (10199812)
• Project Period (FY): 1999 – 2001
• Keywords: Crk / FRET / fluorescent resonance energy transfer / signal transduction / adaptor protein / Ras / Rap1 / biotechnology

Research Abstract

Crk oncogene product activates Ras and Rap1. Ras is a well-known oncogene, whereas Rap1 antagonize Ras-dependent oncogenesis. To understand the mechanism by which Crk activates both oncogene and anti-oncogene, we developed a FRET-based single-molecule probe that can monitor the activation of Crk in a living cell. Using this probe, we visualized rapid Crk activation in an EGF-stimulated cell. However, with the prototype probe, we could not determine where in the cell the activation of Crk starts, due to rapid diffusion of the probe. To overcome this flaw, we fused CAAX box of Ki-Ras protein to the probe, expecting the anchoring of probe to the plasma membrane. Indeed, by the use of this CAAX-fused probe, we found that Crk was activated from the peripheral region of the cell. Furthermore, we generated a recombinant adenovirus for the expression of the probe, which enabled us rapid and efficient expression of the probe in a variety of cells. Thus, the development of new probe for the Crk activity visualized the spatio temporal regulation of Crk oncogene products in living cells and promised us for the understanding of the role of Crk oncogene products in the regulation of cell growth and transformation.

Research Products

• [Publications] K.Kurakawa, et al.: "A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine phosphorylation of the CrkII Adaptor Protein in Vivo."J. Boil Chem.. 276. 31305-31310 (2001)
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• [Publications] K.Kurokawa., N-Mochizuki., Y.Ohba., H.Mizuno., A.Miyawaki., and M.Matsuda: "A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo"J. Biol. Chem.. 276. 31305-31310 (2001)
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• [Publications] Mochizuki, N., Y. Ohba, S. Kobayashi, N. Otsuka, A. M. Graybiel, and <M. Matsuda>__________-: "Crk Activation of JNK via C3G and R-Ras"J. Biol. Chem.. 275. 12667-12671 (2000)
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• [Publications] Yamashita, S., N. Mochizuki, Y. Ohba, M. Tobiume, Y. Okada, H. Sawa, K. Nagashima, and <M. Matsuda>__________-: "CalDAG-GEFIII activation of Ras, R-Ras, and Rap1"J. Biol. Chem.. 275. 25488-25493 (2000)
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• [Publications] Ohba, Y., N. Mochizuki, K. Matsuo, S. Yamashita, M. Nakaya, Y. Hashimoto, M. Hamaguchi, T. Kurata, K. Nagashima, and <M. Matsuda>__________-: "Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade"Mol. Cell. Biol.. 20. 6074-6083 (2000)
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• [Publications] Ohba, Y., N. Mochizuki, S. Yamashita, A. M. Chan, J. W. Schrader, S. Hattori, K. Nagashima, and <M. Matsuda>__________-: "Regulatory proteins of R-ras, TC21/R-Ras2, and M-Ras/R-ras3"J. Biol. Chem.. 275. 20020-20026 (2000)
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