Nuclear Import of Creb and AP-1 Transcription Factors Requires Importin-#beta#_1 and Ran but Is Independent of Importin-#alpha

摘要

Although the specific role of transcription factors (TFs) is nuclear, surprisingly little is known in quantitative terms regarding the pathways by which TFs localize in the nucleus. In this study, we use direct binding assays, native gel electrophoresis, and fluorescence polarization measurements to show for the first time that the cAMP-response element binding protein (CREB) and related AP- 1 and jun and fos constituents are recognized by importin 131 (Impfl) with nanomolar affinity. We reconstitute the nuclear import of these TFs in vitro, demonstrating dependence on cytosolic factors, and show that this is due to the requirement for Imp/3, since antibodies to Imp~3, but not to importin a (Impa), inhibit nuclear accumulation significantly. We show that Imp/3 is necessary and sufficient for docking of CREB at the nuclear envelope; that Ran is essential for CREB nuclear import is demonstrated by the reduction of nuclear accumulation effected by RanGTPyS but not RanGDP, and by dissociation of the Jmp/3—CREB-GFP complex by RanGTPyS but not RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Imp/il- and Ran-mediated nuclear import pathway for CREB and related constitutively nuclear TFs, which is Impa-independent and thus distinct from import pathways utilized by inducible TFs.