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The focus of our study is to identify posttranslational modifications of TRAF2 and characterize their role in TRAF2-mediated activation of JNK and NF-kB signaling pathways. TRAF2 is a key regulatory protein that mediates signals from the TNF receptors, resulting in sequential activation of MAP3K, MAP2K and MAPK (JNK, p38), as well as in activation of RIP/IKK signaling pathways. MAPK and IKK activate AP-1 and NF-kB transcription factors, which in turn induce diverse cellular responses from immune response, proliferation, and differentiation to apoptosis. Although, the signaling mechanisms from RIP/IKK to NF-kB and from MAP3K to AP-1 are better understood, the receptor proximal events that determine TRAF2-dependent activation of RIP/IKK vs. MAP3K remain largely elusive. We have reported that TNFa treatment causes rapid TRAF2 ubiquitination in vivo, and this ubiquitination induces the translocation of TRAF2 to membrane rafts, resulting in selective activation of JNK but not of IKK. Recently, we mapped the phosphorylation and ubiquitination sites of TRAF2. Preliminary experiments indicate that TRAF2 phosphorylation and ubiquitination play critical roles in MAPK and IKK activation in response to TNFa stimulation. We are now systematically investigating the role TRAF2 phosphorylation and ubiquitination in MAPK and IKK activation in response to diverse extracellular stimuli, which will shed a new light on understanding the molecular mechanisms for TRAF2-meditaed activation of MAKP and IKK. Almost all cellular stresses, mammalian oncoproteins and viral transforming proteins activate NF-kB. Activation of NF-kB promotes cellular proliferation and inhibits apoptosis, therefore counteracting p53 and favoring cancer development. Consistent with this, many human tumors display an elevated basal NF-kB activity. In fact, stress- and oncogene-induced NF-kB activation is one of the major obstacles for cancer chemotherapy. However, the mechanism underlying the constitutive activation of NF-kB in human tumors and stress- and oncoprotein-induced NF-kB activation in cancer cells are still elusive. Recently, we identified the kinases that are involved in TRAF2 phosphorylation. Interestingly, these kinases turned out to be well known oncoproteins that are activated by growth factors and diverse cellular stresses, indicating the possibility that TRAF2 phosphorylation by these kinases might be the missing link that regulates oncoprotein- and stress-induced NF-kB activation. We are currently investigating the role of these kinases in TRAF2 phosphorylation and stress-induced NF-kB activation, which will certainly provide mechanistic information for the pathological causal relationship between TRAF2 phosphorylation and increased cancer cell resistance to stress-induced apoptosis. Selected publications: 1. Habelhah H, F. Okada, M. Kobayashi, K. Nakai, S. Choi, J. Hamada, M. Kaya, K. Yoshida, K. Fujinaga, and M. Hosokawa (1999). Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression. Oncogene, 18(9):1771-6. 2. Habelhah H, Shah K, Huang L, Ostareck-Lederer A, Burlingame AL, Shokat KM, Hentze MW and Ronai Z. (2001) ERK phosphorylation drives cotoplasmic accumulation of hnRNP-K and subsequent inhibition of mRNA translation. Nature Cell Biology. 3(3):325-30. 3. Habelhah H, Shah K, Huang L, Burlingame AL, Shokat KM and Ronai Z. (2001) Identification of New JNK substrate using ATP pocket mutant JNK and a corresponding ATP Analogue. J Biol Chem. 276(21):18090-5. 4. Minamoto T, Buschmann T, Habelhah H, Matusevich E, Tahara H, Boerresen-Dale AL, Harris C, Sidransky D, Ronai Z.(2001) Distinct pattern of p53 phosphorylation in human tumors. Oncogene. 20(26):3341-7. 5. Habelhah H, Frew IJ, Laine A, Janes PW, Relaix F, Sassoon D, Bowtell DD, Ronai Z. (2002) Stress-induced decrease in TRAF2 stability is mediated by Siah2. EMBO J. 21(21):5756-65. 6. Habelhah H, Takahashi S, Cho SG, Kadoya T, Watanabe T, and Ronai Z. (2004) Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-kB. EMBO J. 23(2): 322-332. 7. Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, Bhoumik A, Kadoya T, Erdjument-Bromage H, Tempst P. Frapell PB, Bowtell DD and Ronai Z (2004) Siah2 regulates stability of the prolyl-hydroxylases, controls HIF1a abundance and modulates physiological response to hypoxia. Cell. 117:941-952. 8. Habelhah H, Laine A, Erdjument-Bromage H, Tempst P, M. Gershwin E, Bowtell DD and Ronai Z (2004) Regulation of 2-oxoglutarate (a ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah. J Biol Chem. 279(51): 53782-8.
1) Characterize the role of TRAF2 posttranslational modifications in TNFa-induced activation of MAPK and IKK signaling pathways; 2) Explore the molecular mechanism for oncoprotein- and stress-induced NF-kB activation and cancer cell resistance to stress-induced apoptosis; 3) Identify new JNK substrates using ATP analogue and analogue sensitive JNK mutant for in vitro kinase reaction. |
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