Nuclear Import of Creb and AP-1 Transcription Factors Requires Importin-β1 and Ran but Is Independent of Importin-α

Abstract

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 beta1 (Impbeta) 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 Impbeta, since antibodies to Impbeta, but not to importin alpha (Impalpha), inhibit nuclear accumulation significantly. We show that Impbeta 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 RanGTPgammaS but not RanGDP, and by dissociation of the Impbeta-CREB-GFP complex by RanGTPgammaS but not RanGDP as demonstrated using fluorescence polarization assays. The results support the existence of an Impbeta1- and Ran-mediated nuclear import pathway for CREB and related constitutively nuclear TFs, which is Impalpha-independent and thus distinct from import pathways utilized by inducible TFs.

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

“…Similar to the cNLS, the HMG domain was recognized poorly by IMP␣ (K d ϭ 112 nM), although IMP␤ recognition was slightly increased in the presence of IMP␣ (K d ϭ 2.4 nM). Thus, SRY joins a less common but growing list of arginine-rich NLScontaining proteins recognized by IMP␤ rather than IMP␣ (14,22,29,(31)(32)(33) that include the Rev (RqaRRnRRRRwR) from HIV type 1 (32, 34), the Rex protein of human T cell leukemia virus type 1 (mpKtRRRpRRsqRKRppt) (33), and the AP-1 family transcription factor CREB (26).…”

“…Mouse IMP␣-GST and IMP␤-GST were expressed as described (24). Their functionality in terms of mediating nuclear import has been demonstrated (25,26).…”

Transportin Is a Major Nuclear Import Receptor for c-Fos

“…Nuclear transport of c-Fos and c-Jun appears to be independent of importin ␣, because both proteins interact directly with the transport receptor importin ␤. The apparent affinities of the transcription factors for importin ␤, however, are rather low (13), and importin ␤-dependent transport has not been demonstrated in a permeabilized cell system so far.…”

“…Some of these bind directly to the classic import receptor, importin ␤, without requiring any adapter protein. These include the proteins Rev and Rex of the human immunodeficiency virus (5,6) and the human T-cell leukemia virus (7), respectively, the T-cell protein tyrosine phosphatase (8), the parathyroid hormone-related protein (PTHrP) (9), cyclin B1 (10), the sterol regulatory element-binding protein 2 (SREBP-2 (11)), the zinc finger protein Snail (12), and the transcription factor CREB (13). Many of these proteins also contain basic NLSs.…”

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

“…The cargo⅐importin-␣/-␤ complex is transported into the nucleus through the nuclear pore complex (NPC) (18,20). Several recent data have demonstrated that certain proteins are transported into the nucleus by importin-␣ alone (21), or by importin-␤ alone (22)(23)(24)(25)(26)(27).…”