Primary Study On Molecular Mechanism Of STAT4Nuclear Import

Nuclear import is the key premise for nuclear transcription factors to transduce signal transduction. It is found that Signal Transducers and activators of transcription (STATs) do not own nuclear location signal (NLS) which can conduct protein nuclear import. However, STATs can be transiently accumulated in nuclear after stimulated by some cytokines. Therefore, study on the mechanism of STATs nuclear translocation is very interesting. It was reported that a certain sequence in the DNA binding domain of STAT1as well as STAT3promotes its entry into the nuclear. Whether a homologous sequence in the DNA binding domain of STAT4which can induce STAT4nuclear import is not reported at present.Objective:To explore the mechanism of STAT4nuclear import stimulated by IL-12.Methods:1. Align the DNA binding domain of STAT1. STAT3with other STATs in order to find the homologous sequence in STAT4assayed by bioinformatics.2. Several plasmids were constructed by molecular subcloning technique. Wild-type STAT4was inserted into pEGFP-Cl to construct pEGFP-STAT4. pEGFP-STAT4-Del was constructed by inserting STAT4deleted amino acids395-416. Classic NLS DNA sequence of SV40T antigen was inserted into pEGFP-Cl vector used as a positive control (pEGFP-NLS). STAT4-Del fragment was inserted into pEGFP-NLS and this plasmid was named pEGFP-NLS-STAT4-Del. These plasmids were confirmed by restriction enzymes digestion and DNA sequencing. The expression of these plasmids transfected in eukaryotic cells was checked by immunoblot.3. Above plasmids were transiently transfected into Caski cells. After cells were stimulated by IL-12, the positive signals in the transfected cells were observed by fluorescence microscopy.Results:1. It was found that amino acids395-416in STAT4is a potential dimer-specific NLS (dsNLS) analyzed by alignment of DNA binding domain in STATs.2. The constructed plasmids were confirmed by restriction enzyme digestion and DNA sequencing. These plasmids were expressed correctly in Caski cells detected by transfection and immunoblot.3. pEGFP-STAT4and pEGFP-STAT4-Del were transfected into cells, respectively. After the transfected cells were stimulated by IL-12, it was found that wild type STAT4was imported into nuclear, while STAT4-Del was retarded in cytoplasm. Furthermore, treated the transfected cells with Leptomycin (LMB), following IL-12 stimulation again, it was found that wild type STAT4was located in cytoplasm at Omin, then translocated into nuclear at45min, and was still kept in nuclear at60min. However, STAT4-Del was kept in cytoplasm with above treatments. Additionally, insertion of classic NLS into EGFP-STAT4-Del restored nuclear import of STAT4-Del.Conclusion:The sequence, amino acid395-416of STAT4, is a dsNLS, mediating STAT4nuclear import after stimulation by IL-12.