|Budget Amount *help
¥8,400,000 (Direct Cost : ¥8,400,000)
Fiscal Year 1997 : ¥3,700,000 (Direct Cost : ¥3,700,000)
Fiscal Year 1996 : ¥4,700,000 (Direct Cost : ¥4,700,000)
Thus far, extensive studies have been mainly concentrated on developing an understanding of the molecular mechanism of unclear import of SV40 T-NLS (nuclear localization signal) containing substrate, and as a result, many significant findings have been obtained. The SV40 T-antigen is a good candidate for karyophilic proteins which are transported constitutively and immediately after synthesis on free ribosomes in the cytoplasm. However, how proteins, which preexist in the cytoplasm at steady state, migrate into the nucleus in response to extracellular signal, remains unknown. To answer this question, we used a transcription factor, aSTAT (signal transducers and activators of transcription) protein, as a model substrate. In response to interferon-gamma (IFN-gamma), STAT1 is tyrosine phosphorylated and translocates to the nucleus. In this study, we found that tyrosine-phosphorylated STAT1 associated with the beta subunit of the nuclear pore-targeting complex via the NPI-1 family, but not the Rch 1 family, of the alpha subunit. Antibodies against NPI-1 or beta subunit inhibited the IFN-gamma-dependent nuclear import of STAT1 in living cells. Solution binding assays with deletion mutants of NPI-1 showed that the STAT1-binding domain of NPI-1 was located in the carboxy-terminal region, which is clearly distinct from the SV40 T-NLS.These results indicate that the extracellular signal-dependent nuclear transport of STAT1 is mediated by NPI-1, but not Rch1, in conjunction with beta subunit. Moreover, we found that nuclear import of STAT1 was suppressed by microinjection of the antibody against a small GTPase, Ran, and two mutant Ran proteins, one defective in GTP hydrolysis (G19V) and the other with little or no binding to GTP (T24N), both of which are known to act as dominant negative inhibitors of nuclear import. These results indicate that the conditional nuclear import of STAT1 requires Ran.