Nuclear Import Mechanisms of STAT and NF-κB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs), large protein complexes that penetrate and fuse the nuclear envelope. NPCs are freely permeable to small molecules (such as metabolites and water) but they restrict the movement of larger molecules to those containing specific transport signals. Proteins to be targeted into the nucleus by the classical nuclear import system contain arginine/lysine-rich nuclear localization signals (NLSs), which are recognized by importin α, the NLS receptor. Importin α binds to importin β, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin α isoforms have been identified: importin α1, α3, α4, α5, α6 and α7. Many intracellular signal transduction pathways are regulated by controlling the nuclear localization of specific proteins. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes. These sequences are usually located in the promoter regions preceding the gene, and the binding of transcription factors to these sites modulates the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor κB (NF-κB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-κB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-κB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin α5 molecule via unconventional dimer-specific NLSs. The results of this work further suggest that two of these NLS elements, one in each monomer, have to be intact for STAT1 homodimers and STAT1/STAT2 heterodimers to be recognizable by importin α5 and subsequent nuclear import to take place. Moreover, we found that importin α5STAT complex is composed of one STAT dimer and two importin α5 molecules. Importin α molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin α5 molecule. The results in this work are the first to demonstrate interactions between importin α molecules and NF-κB proteins. This work demonstrates that all NF-κB proteins are transported into the nucleus by importin α molecules. In addition, NLS was identified in RelB protein. The interactions between NF-κB proteins and importin α molecules were found to be directly mediated by the NLSs of NF-κB proteins. Furthermore, this study shows that the classical and the alternative NF-κB pathway components have somewhat different specificities for importin α isoforms. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3. The results in this work show that the nuclear import of p52/RelB dimers is exclusively mediated by the NLS of RelB protein, whereas the nuclear import of p52/p65 dimers is solely regulated by the NLS of p52 protein. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-κB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.